PROJECT SUMMARY 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase isoform 3 (PFKFB3) is a critical glycolytic regulator in vascular cells. PFKFB3 catalyzes the synthesis of fructose-2, 6-bisphosphate (F2, 6P2) and the latter is the most potent activator for 6-phosphofructo-1-kinase (PFK-1), one of three rate-limiting enzymes for glycolysis. Proliferation of vascular cells including endothelial cells and vascular smooth muscle cells is the major feature of vascular remodeling in a variety of vascular diseases including pulmonary arterial hypertension (PAH). Ours and the work from others have demonstrated a critical role of endothelial PFKFB3 in angiogenesis. In our recent studies we have found the following: (1) PDGF/VEGF and hypoxia increase the protein level and activity of PFKFB3 in pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs); (2) inhibition of PFKFB3 attenuates PDGF/VEGF- and hypoxia-induced collagen synthesis, hyperproliferation and resistance to apoptosis of PASMCs and PAECs; (3) hypoxia-induced pulmonary vascular remodeling was significantly suppressed in PFKFB3-/+ mice; (4) PFKFB3 protein levels are robustly increased in the smooth muscle cells and endothelial cells as well as plexiform lesions of pulmonary arteries of patients with idiopathic PAH. These data led us to hypothesize that PFKFB3 in vascular cells has a central role in vascular remodeling and suppression of PFKFB3 inhibits proliferation of vascular cells and further curbs the development of pulmonary arterial hypertension. The goal of this proposal is designed to use specific PFKFB3 inhibitor and tissue-specific PFKFB3 deletion mice to investigate whether and how PFKFB3 in vascular cells plays a crucial role in vascular remodeling of PAH. In Aim 1, we will investigate whether PDGF/VEGF and hypoxia increase PFKFB3 activity via HIF1?-mediated PFKFB3 gene expression and MAPK-mediated PFKFB3 phosphorylation in PASMCs and PAECs. In Aim 2, we will study whether and how lactate, arising from high PFKFB3 activity, elevates Akt activation, leading to collagen synthesis, hyperproliferation and resistance to apoptosis of PASMCs and PAECs. In Aim 3, we will examine whether pulmonary vascular remodeling is suppressed in mice with PFKFB3 deficiency in smooth muscle cells or endothelial cells, and in rat treated with a specific PFKFB3 inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2- propen-1-one (3PO). This proposal is highly translational. Completion of this project will advance a new PFKFB3-based paradigm for the suppression of vascular remodeling and may lead to a more effective and specific therapy of diseases associated with vascular remodeling.